Hepatitis B virus (HBV) chronically infects ~350 million people and causes a million deaths yearly, but there are no widely effective therapies for it. Development of therapies has been hindered by poor understanding of viral replication. We are pursuing a molecular analysis of the duck hepatitis B virus polymerase as a model for the HBV polymerase. We will perform a detailed structure-function analysis of the polymerase using a unique collection of monoclonal antibodies along with active recombinant polymerase and polymerase from virions and infected duck liver.Aim 1. How is polymerase translation initiated? Expression of the core and polymerase proteins is closely linked because they are translated from a bicistronic mRNA. We recently demonstrated that the polymerase is translated more rapidly than is core despite its downstream position on the mRNA. We will determine the mechanism(s) for the unusually high translation rate of the polymerase to begin to understand how this may contribute to the viral replication cycle.Aim 2. What is the size and multimeric state of the polymerase in virions and cells? The polymerase is believed to be a monomeric, uncleaved protein. However, we hypothesize that its structure is more complex because activity gel analysis detects species larger than expected and polymerase molecules smaller than its predicted mass can be detected by western analysis of cell lysates. We will employ our unique antibodies to determine these structural parameters.Aim 3. Which amino acids of the polymerase are involved in the early stages of reverse transcription? Knowing these sequences would be of value in rational design of inhibitors of the polymerase, just as identification of key residues of the HIV protease assisted development of inhibitors. We will therefore map residues involved in reverse transcription with anti-polymerase antibodies, mutagenesis, and UV cross-linking.The proposed studies will fill basic gaps in our knowledge of hepadnaviral replication and will guide design of antiviral therapies.